Peptides with vasodilatory and/or diuretic functions

ABSTRACT

The present invention relates to peptides with vasodilatory and/or diuretic functions. In particular, the invention relates to modifying key amino acid residues in natriuretic peptides to achieve different functions and properties. Accordingly the invention also includes modified natriuretic peptides. The invention also relates to the use of these peptides for regulating blood pressure-volume and/or treating a heart condition.

FIELD OF THE INVENTION

The present invention relates to peptides with vasodilatory and/ordiuretic functions. The invention also relates to the use of thesepeptides for regulating blood pressure-volume.

BACKGROUND OF THE INVENTION

Heart failure (HF) is one of the leading causes of death in the world (1in 4 deaths) [Roger, V. L.; et al., Circulation 123 (4): e18-e209(2011)]. Although several factors such as coronary artery disease,myocardial infarction, prolonged hypertension, cardiomyopathy couldinitiate HF, the progression of the syndrome is linked to the activationof neurohumoral systems, specifically of that of (renin-angiotensinaldosterone system (RAAS) and sympathetic (SNS) which elevate bloodvolume and pressure [MacMahon, K. M.; Lip, G. Y., Arch Intern Med 162(5): 509-16 (2002)]. The distinctive features of HF encompass functionaland structural changes in the heart along with vasoconstriction, avidretention of water and sodium by kidney to various degrees [McMurray, J.J.; Pfeffer, M. A., Lancet 365 (9474): 1877-89 (2005)]. The patientswith HF may be broadly classified into: (a) hypertensive HF witheuvolemic or mildly hypervolemic, (b) normotensive HF with hypovolemicand (c) hypotensive HF with hypervolemic or hypovolemic states [Packer,M., Am J Cardiol 71 (9): 3C-11C (1993); Strobeck, J. E.; Silver, M. A.,Congest Heart Fail 10 (2 Suppl 2): 1-6 (2004)].

Natriuretic peptides (NPs) are a class of vital hormones which confercardiovascular protection through regulation of vascular tone and fluidvolume in the body [Brenner, B. M.; et al., Physiol Rev 70 (3): 665-99(1990)]. They have indispensable roles in influencing hemodynamics of anorganism under physiological and pathological conditions. There arethree isoforms of NPs—ANP, BNP and CNP, identified in mammals [Kangawa,K.; Matsuo, H., Biochem Biophys Res Commun 118 (1): 131-9 (1984); Sudoh,T.; et al., Nature 332 (6159): 78-81 (1988); Sudoh, T.; et al., BiochemBiophys Res Commun 168 (2): 863-70 (1990)]. These peptides have anevolutionarily conserved 17-residue ring held by a disulphide bond and avariable N- and C-terminal tails. ANP and BNP are secreted by thecardiac walls in response to increasing filling pressures in thechambers of the heart [de Bold, A. J., Science 230 (4727): 767-70(1985)] while CNP is derived from vascular endothelium [Suga, S.; etal., Endocrinology 133 (6): 3038-41 (1993)]. These peptides bind tospecific membrane bound natriuretic peptide receptors (NPR-A, NPR-B orNPR-C). ANP and BNP activate NPR-A [Waldman, S. A.; et al., J Biol Chem259 (23): 14332-4 (1984)] and CNP functions through NPR-B to elevateintracellular cGMP levels [Suga, S.; et al., Endocrinology 130 (1):229-39 (1992)]. The downstream activities of ANP/NPR-A signaling includevasodilation, increased excretion of water and electrolytes throughkidneys, increased endothelial permeability and inhibition ofrenin-angiotensin aldosterone system (RAAS) and sympathetic nervoussystem (SNS) [Brenner, B. M.; et al., Physiol Rev 70 (3): 665-99 (1990);Maack, T.; et al., Fed Proc 45 (7): 2128-32 (1986); Sasaki, A.; et al.,Eur J Pharmacol 109 (3): 405-7 (1985)]. These direct effects onhemodynamics and the inhibition of secretion of antagonistic factorscontribute to the antihypertensive and antihypervolemic action of NPs[Brenner, B. M.; et al., Physiol Rev 70 (3): 665-99 (1990)]. Incontrast, CNP/NPR-B signaling is mainly involved in tissue remodeling,reproduction and brain functions along with mild hypotensive effectsthrough vasodilation [Chusho, H.; et al., Proc Natl Acad Sci USA 98 (7):4016-21 (2001)]. The residues within the conserved NP ring are necessaryfor receptor binding while the molecular recognition to specific NPRsare due to subtle differences in their sequences such as C-terminalextensions [Brenner, B. M.; et al., Physiol Rev 70 (3): 665-99 (1990)].

The present treatment strategies for HF include the pharmacologicalintervention of RAAS and SNS activation using angiotensin convertingenzyme inhibitors, neprilysin inhibitors, angiotensin receptorinhibitors and diuretics which reduce blood volume and pressure andaugment the NP activity and cGMP production as the secondary response[Rogers, C.; Bush, N., Nurs Clin North Am 50 (4): 787-99 (2015)]. NPshave a crucial role in restoring the pressure-volume homeostasis throughregulation of vascular tone, natriuresis, diuresis and inhibition ofRAAS and SNS [Lee, C. Y.; Burnett, J. C., Jr., Heart Fail Rev 12 (2):131-42 (2007)]. Both ANP and BNP levels are elevated in patients withHF, but they fail to exhibit their beneficiary roles due to (a) loweravailability of bio-active peptide and (b) lower expression level of theNP receptors [Mukaddam-Daher, S., Expert Opin Ther Targets 10 (2):239-52 (2006)]. Despite their elevation, exogenous infusion of ANP orBNP in HF patients has shown to improve the clinical status of thepatients [Lee, C. Y.; Burnett, J. C., Jr., Heart Fail Rev 12 (2): 131-42(2007); Mukaddam-Daher, S., Expert Opin Ther Targets 10 (2): 239-52(2006)]. Despite the beneficiary roles, the exogenous infusion of ANPand BNP has been associated to cause distress due to their diversephysiological actions. Hence, ligands that differentiate the vascularand renal functions of NPs would be of great value to address thepersonalized needs of various HF patients with distinct imbalances[Strobeck, J. E.; Silver, M. A., Congest Heart Fail 10 (2 Suppl 2): 1-6(2004); Volpe, M.; et al., Clin Sci (Lond) 130 (2): 57-77 (2016)].

HF may be broadly classified into: (a) hypertensive HF with euvolemic ormildly hypervolemic, (b) normotensive HF with hypovolemic and (c)hypotensive HF with hypervolemic or hypovolemic states. Natriureticpeptides (NPs) are key hormones which lower blood pressure and volumethrough their multifaceted actions on blood vessels, kidney, heart andsympathetic nervous system. Infusion of NPs in patients with HF has beenbeneficial due to their multifaceted action in reducing pressure-volumeoverload. However, NPs infusion has also been associated with severehypotension.

New drug leads for the different tensive and volemic states in heartfailure are desirable.

SUMMARY OF THE INVENTION

The present invention provides peptides which modulate blood pressure byhaving vasodilatory and/or diuretic activity.

According to a first aspect, the present invention provides an isolatedpeptide comprising SEQ ID NO: 1 (CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC), whereinX₁, X₂, X₈ each independently comprises any amino acid; X₃ comprises Ror K; X₄ comprises M, I or L; X₅ comprises G, S or N; X₆ comprises A, Hor S; X₇ comprises Q, T, S, M or V; and X₉ comprises I or L.

According to a second aspect, the present invention provides an isolatedpeptide according to any aspect of the present invention for use inmedicine; for use in therapy; and/or for use as a medicament.

According to a third aspect, the present invention provides the use ofat least one isolated peptide according to any aspect of the presentinvention in the preparation of a medicament for regulating bloodpressure-volume and/or for treating a heart condition.

According to a fourth aspect, the present invention provides a method ofregulating blood pressure-volume and/or treating a heart condition in asubject comprising administering an effective amount of an isolatedpeptide according to any according to any aspect of the presentinvention to the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show K-Ring is a vasodilator. FIG. 1A: Sequence comparisonof mammalian and venom NPs. NPs have a canonical 17-membered ring heldby a disulphide bond. Residues in pink are conserved among all NPs andresidues highlighted in shade are important for NP receptor binding.K-Ring are two key substitutions at position 3 and 14, conserved Gresidues are replaced by D, and it has a two residue C-terminal tail.Male SD rats were anesthetized with sodium pentobarbital and theirfemoral artery and vein and urinary bladder was catheterized. Salinewith or without the peptide was infused all though the experiment.Shaded region represents the infusion of the peptide. FIG. 1B:Dose-dependent effect of ANP and K-Ring on MAP of anesthetized rats.FIG. 1C: Dose-dependent effects of ANP and K-Ring on urine flow rate inanesthetized rats. The data are represented as mean±SE of fiveindependent experiments.

Control group, 0.2 nmol/kg/min ANP and 2 nmol/kg/min K-Ring profiles areadapted from Sridharan et al., [Biochem J 469 (2): 255-66 (2015)].

FIGS. 2A-2C show Aspartate residues in the ring and the C-terminal tailare molecular switches that control in-vivo activities of NPs. FIG. 2A:Sequence of K-Ring variants and their in vivo response (Substitutedresidues are shown in green). Femoral artery, vein and urinary bladderof male SD rats anesthetized with sodium pentobarbital were catheterizedto measure hemodynamic and urine volumes in line with the infusion ofthe peptides. Saline with or without peptide was continuously infusedthroughout the experiment. Shaded region represents the period ofinfusion of the peptide. FIG. 2B: Dose-dependent (2 nmol/kg/min) effectof K-Ring and its variants on MAP of anesthetized rats. FIG. 2C:Dose-dependent effects of K-Ring and its variants on urine flow rate inanesthetized rats. The data are represented as mean±SE of fiveindependent experiments.

Control and 2 nmol/kg/min K-Ring profiles have been adapted fromSridharan et al., [Biochem J 469 (2): 255-66 (2015)].

FIGS. 3A-3C show incorporation of asparate and arginine residues in ANPscaffold alters vascular and renal functions. FIG. 3A: Sequence of ANPvariants and their in vivo response (Substituted residues are shown ingreen). Femoral artery, vein and urinary bladder of male SD ratsanesthetized with sodium pentobarbital were catheterized to measurehemodynamic and urine volumes in line with the infusion of the peptides.Saline with or without peptide was continuously infused throughout theexperiment. Shaded region represents the period of infusion of thepeptide. FIG. 3B: Dose-dependent effect of ANP and its variants on MAPof anesthetized rats. FIG. 3C: Dose dependent effects of ANP and itsvariants on urine flow rate in anesthetized rats. The data arerepresented as mean±SE of five independent experiments.

Control and 0.2 nmol/kg/min ANP profiles have been adapted fromSridharan et al., [Biochem J 469 (2): 255-66 (2015)].

FIGS. 4A-4F show variants of ANP and K-Ring are partial agonists ofNPR-A. Amount of cGMP accumulated after the treatment of cells(transiently expressing NPR-A) with NP analogues were determined. FIGS.4A, 4B: Dose-response of cGMP production after 30 min treatment withANP, K-Ring and their variants. FIGS. 4C, 4D: Activation kinetics ofNPR-A measured through time-chase study using EC 50 concentration ofpeptides. FIGS. 4E, 4F: Activation kinetics of NPR-A per pmole of NPs.The data are represented as mean±SE of three independent experiments.

FIG. 5 shows molecular mass and homogeneity of ANP, K-Ring and theirvariants. Synthetic ANP, K-Ring and their variants were synthesizedusing Fmoc-based solid phase peptide chemistry and purified byreversed-phase HPLC. The purified peptides were oxidized using 10% DMSOsolution under alkaline conditions and purified HPLC. The mass of thepurified, oxidized peptides were analyzed by ESI-Ion trap MS. Calculatedmasses of peptides are shown.

FIGS. 6A-6D show changes in vascular parameters in response to dosedependent infusion of ANP and K-Ring.

Femoral artery, vein and urinary bladder were catheterized in male SDrats anesthetized using sodium pentobarbital. Saline with or without thepeptides was infused continuously until the end of experiment. Shadedregion represents the fusion of peptide. FIG. 6A: Heart rate, FIG. 6B:Pulse pressure, FIG. 6C: Systolic pressure, FIG. 6D: Diastolic pressurechanges associated with infusion of two different doses of ANP andK-Ring.

FIGS. 7A-7D show changes in vascular parameters in response to dosedependent infusion of K-Ring variants.

Femoral artery, vein and urinary bladder were catheterized in male SDrats anesthetized using sodium pentobarbital. Saline with or without thepeptides was infused continuously until the end of experiment. Shadedregion represents the fusion of peptide. FIG. 7A: Heart rate, FIG. 7B:Pulse pressure, FIG. 7C: Systolic pressure, FIG. 7D: Diastolic pressurechanges associated with infusion of K-Ring variants.Data shown are an average of five independent trails and represented asmean±SE. Control and 2 nmol/kg/min K-Ring profiles have been adaptedfrom Sridharan et al., [Biochem J 469 (2): 255-66 (2015)].

FIGS. 8A-8D show changes in vascular parameters in response to dosedependent infusion of ANP variants.

Femoral artery, vein and urinary bladder were catheterized in male SDrats anesthetized using sodium pentobarbital. Saline with or without thepeptides was infused continuously until the end of experiment. Shadedregion represents the fusion of peptide. FIG. 8A: Heart rate, FIG. 8B:Pulse pressure, FIG. 8C: Systolic pressure, FIG. 8D: Diastolic pressurechanges associated with infusion of ANP variants.Data shown are an average of five independent trails and represented asmean±SE. Control group, 0.2 nmol/kg/min ANP and 2 nmol/kg/min K-Ringprofiles are adapted from Sridharan et al., [Biochem J 469 (2): 255-66(2015)].

DEFINITIONS

Certain terms employed in the specification, examples and appendedclaims are collected here for convenience.

The term “amino acid sequence,” as used herein, refers to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, an aminoacid residue that is substituted at position 2, 3 and/or 14 of the17-residue ring of the natriuretic peptide to effect a change in peptideactivity could itself be replaced by a similar amino acid withoutsignificantly altering the activity. For example, if D (Asp) issubstituted into the 17-residue ring of the natriuretic peptide toeffect a change in peptide activity, it could be replaced with anotheramino acid residue from the same (acidic residue) side chain family suchas E (Glu). Likewise, K (Lys) could be used instead of R (Arg), as bothare basic residues. Moreover, the C-terminal NSFRY is known to interactwith the receptor. Amino acid Q (Gin) is equivalent to N (Asn) (bothhydrophilic, neutral residues), T (Thr) is equivalent to S (Ser) (bothhydrophilic, neutral residues with hydroxyl side chains), and Y (Tyr)and W (Trp) are equivalent to F (Phe) (both hydrophobic, aromaticresidues). Permutations of these residues may also help in modifying theC-terminal tail.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence of the invention. The compositionmay comprise a dry formulation or an aqueous solution.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material.

The term “subject” is herein defined as vertebrate, particularly mammal,more particularly human. For purposes of research, the subject mayparticularly be at least one animal model, e.g., a mouse, rat and thelike. In particular, for treatment or prophylaxis of bloodpressure-related diseases, the subject may be a human.

The term “treatment”, as used in the context of the invention refers toameliorating, therapeutic or curative treatment.

As used herein, the term “comprising” or “including” is to beinterpreted as specifying the presence of the stated features, integers,steps or components as referred to, but does not preclude the presenceor addition of one or more features, integers, steps or components, orgroups thereof. However, in context with the present disclosure, theterm “comprising” or “including” also includes “consisting of”. Thevariations of the word “comprising”, such as “comprise” and “comprises”,and “including”, such as “include” and “includes”, have correspondinglyvaried meanings.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are forconvenience listed in the form of a list of references and added at theend of the examples.

The present invention provides an isolated peptide comprising SEQ ID NO:1 (CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC), wherein X₁, X₂, X₈ each independentlycomprises any amino acid; X₃ comprises R or K; X₄ comprises M, I or L;X₅ comprises G, S or N; X₆ comprises A, H or S; X₇ comprises Q, T, S, Mor V; and X₉ comprises I or L.

In one preferred embodiment, the isolated peptides and/or nucleic acidof the invention exclude a naturally occurring peptide and/or a nucleicacid molecule, unless they are modified in some way by standard methodsknown in the art for the purpose of, for example, increasing theirstability in vivo and/or in vitro.

In another preferred embodiment, an isolated cDNA molecule whichreplicates a naturally occurring sequence of exons encoding naturallyoccurring peptide is also excluded from the present invention.

Examples of naturally occurring peptides are:

Atrial natriuretic peptide (ANP) with sequence:

(SEQ ID NO: 52) SLRRSSCFGGRMDRIGAQSGLGCNSFRYKrait venom natriuretic peptide (KNP) with sequence:

(SEQ ID NO: 53) GLLISCFDRRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAAB-type natriuretic peptide (BNP) with sequence:

(SEQ ID NO: 54) SPKMVQGSGCFGRKMDRISSSSGLGCKVLRRHC-type natriuretic peptide (CNP) with sequence:

(SEQ ID NO: 55) GLSKGCFGLKLDRIGSMSGLGCDendroaspis natriuretic peptide (DNP) with sequence:

(SEQ ID NO: 56) EVKYDPCFGHKIDRINHVSNLGCPSLRDPRPNAPSTSA

Any use of a cDNA molecule; a naturally occurring peptide and/or anaturally occurring nucleic acid molecule will be regarded as part ofthe present invention.

It will be appreciated that the present invention includes an isolatedpeptide comprising or consisting of a sequence of the 17-residue ring ofa natriuretic peptide.

It will be appreciated that the present invention also includes anisolated peptide comprising a sequence of the 17-residue ring of anatriuretic peptide comprising at least one modified amino acid atpositions 2, 3 and/or 14 of the 17-residue ring of the natriureticpeptide. By “modified” the term is intended to include substitution ofone amino acid by another in the 17-residue ring of a natriureticpeptide. For example, amino acid G (Gly) may replace amino acid D (Asp)at position 2.

It will be further appreciated that the present invention includes anisolated full-length natriuretic peptide comprising at least onemodified amino acid at positions 2, 3 and/or 14 of its 17-residue ring.Moreover, it would be understood that an amino acid substitution made atthe position 2, 3 and/or 14 could be replaced by a conservative aminoacid. For example, if R (Arg) is substituted into position 3 anothersimilar (conservative) amino acid could replicate the effect of the R onpeptide activity; such as substitution with K (Lys).

The present invention is further described by the following embodimentsof invention.

According to a first aspect, the present invention provides an isolatedpeptide having vasodilatory and/or diuretic activity in mammals,comprising the amino acid sequence SEQ ID NO: 1(CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC), wherein X₁, X₂, X₈ each independentlycomprises any amino acid; X₃ comprises R or K; X₄ comprises M, I or L;X₅ comprises G, S or N; X₆ comprises A, H or S; X₇ comprises Q, T, S, Mor V; and X₉ comprises I or L, wherein the peptide does not consist ofthe amino acid sequence depicted by SEQ ID NO: 52, SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 55 or SEQ ID NO: 56.

According to a preferred embodiment the isolated peptide according tothe first aspect comprises SEQ ID NO: 2 (CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC)wherein X₁ comprises D or G. X₁ could comprise E instead of D.

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 3 (CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC)wherein X₂ comprises R or G. X₂ could comprise K instead of R.

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 4 (CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC),wherein X₈ comprises D or G. X₈ could comprise E instead of D.

According to another preferred embodiment the isolated peptide accordingto the first aspect or first preferred embodiment comprises SEQ ID NO: 5(CFDX₂X₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect or first preferred embodiment comprises SEQ ID NO: 6(CFGX₂X₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect or second preferred embodiment comprises SEQ ID NO:7 (CFX₁RX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect or second preferred embodiment comprises SEQ ID NO:8 (CFX₁GX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect or third preferred embodiment comprises SEQ ID NO: 9(CFX₁X₂X₃X₄DRIX₅X₆X₇SDX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect or third preferred embodiment comprises SEQ ID NO:10 (CFX₁X₂X₃X₄DRIX₅X₆X₇SGX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 11 (CFDRX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 12 (CFGRX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 13 (CFDGX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 14 (CFGGX₃X₄DRIX₅X₆X₇SX₈X₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 15 (CFDX₂X₃X₄DRIX₅X₆X₇SDX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 16 (CFGX₂X₃X₄DRIX₅X₆X₇SDX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 17 (CFDX₂X₃X₄DRIX₅X₆X₇SGX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 18 (CFGX₂X₃X₄DRIX₅X₆X₇SGX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 19 (CFX₁RX₃X₄DRIX₅X₆X₇SDX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 20 (CFX₁RX₃X₄DRIX₅X₆X₇SGX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 21 (CFX₁GX₃X₄DRIX₅X₆X₇SDX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 22 (CFX₁GX₃X₄DRIX₅X₆X₇SGX₉GC).

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 1, wherein X₁ is G or D, X₂ isG or R and X₈ is G or D.

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 1, wherein X₁, X₂ and X₈ areselected from, respectively, G, R and D; D, R and G; D, G and D; and D,R and D.

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises SEQ ID NO: 23(CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GCNSFRY). It would be understood that withinthe NSFRY tail the amino acid N is equivalent to Q (both hydrophilic,neutral residues), S is equivalent to T (both hydrophilic, neutralresidues with hydroxyl side chains), and F is equivalent to W and Y(both hydrophobic, aromatic residues). Substitution with theseconservative residues should not significantly change the activity ofthe C-terminal tail.

According to another preferred embodiment the isolated peptide accordingto the first aspect comprises a sequence selected from the groupconsisting of:

SEQ ID NO: 24 (CFDRRIDRISHTSDIGC); SEQ ID NO: 25 (CFGRRIDRISHTSDIGC);SEQ ID NO: 26 (CFDGRIDRISHTSDIGC); SEQ ID NO: 27 (CFDRRIDRISHTSGIGC);SEQ ID NO: 28 (CFGGRIDRISHTSDIGC); SEQ ID NO: 29 (CFGRRIDRISHTSGIGC);SEQ ID NO: 30 (CFDGRIDRISHTSGIGC); and SEQ ID NO: 31(CFGGRIDRISHTSGIGC).

According to another preferred embodiment the isolated peptide accordingto the first aspect is selected from the group consisting of:

SEQ ID NO: 32 (GLLISCFDRRIDRISHTSDIGCRH); SEQ ID NO: 33(GLLISCFDRRIDRISHTSGIGCRH); SEQ ID NO: 34 (GLLISCFGRRIDRISHTSDIGCRH);SEQ ID NO: 35 (GLLISCFDGRIDRISHTSDIGCRH); SEQ ID NO: 36(GLLISCFGRRIDRISHTSGIGCRH); SEQ ID NO: 37 (GLLISCFDRRIDRISHTSDIGCNSFRY);SEQ ID NO: 38 (GLLISCFGRRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA); SEQ ID NO: 39(GLLISCFDGRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRD LRADSKQSRAA); SEQ IDNO: 40 (GLLISCFDRRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRD LRADSKQSRAA);SEQ ID NO: 41 (GLLISCFGGRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA); SEQ ID NO: 42(GLLISCFGRRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRD LRADSKQSRAA); SEQ IDNO: 43 (GLLISCFDGRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRD LRADSKQSRAA);and SEQ ID NO: 44 (GLLISCFGGRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA).

According to another preferred embodiment the isolated peptide accordingto the first aspect is selected from the group consisting of:

SEQ ID NO: 45 (SLRRSSCFDGRMERIGAQSGLGCNSFRY); SEQ ID NO: 46(SLRRSSCFGRRMDRIGAQSGLGCNSFRY); SEQ ID NO: 47(SLRRSSCFGGRMDRIGAQSDLGCNSFRY); SEQ ID NO: 48(SLRRSSCFDRRMDRIGAQSGLGCNSFRY); SEQ ID NO: 49(SLRRSSCFDGRMDRIGAQSDLGCNSFRY); SEQ ID NO: 50(SLRRSSCFGRRMDRIGAQSDLGCNSFRY); and SEQ ID NO: 51(SLRRSSCFDRRMDRIGAQSDLGCNSFRY).

According to another preferred embodiment there is provided an isolatedpeptide according to the first aspect, wherein when X1 is G and X8 is G,or when NSFRY is present at the C-terminal end of the peptide, diureticactivity of the peptide is elicited.

According to another preferred embodiment there is provided an isolatedpeptide according to the first aspect, wherein

-   -   a) when X1 is G the peptide elicits reduced heart rate and pulse        pressure compared to when X1 is D;    -   b) when X2 is G the peptide elicits exclusive diuresis at low        dose and hypotension along with diuresis at higher dose;    -   c) when X2 is R the peptide elicits a reversed preference of        pharmacological activity to that shown in b);    -   d) when X8 is G the peptide elicits sustained vasodilatory        effects compared to when X8 is D; and    -   e) when X1 is D and X8 is D the peptide elicits exclusive        hypotension without significant diuresis.

In a preferred embodiment of the invention there is provided an isolatedpeptide having vasodilatory and/or diuretic functions. Preferably, theisolated peptide has an amino acid sequence according to any one of SEQID NO: 1 to SEQ ID NO: 51.

According to another preferred embodiment there is provided an isolatedpeptide according to the first aspect, wherein peptides from the groupcomprising SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 51 have vasodilatoryactivity; peptides from the group comprising SEQ ID NO: 29, SEQ ID NO:36 and SEQ ID NO: 37 have vasodilatory and diuretic activity; andpeptides from the group comprising SEQ ID NO: 49, have renal activity.

The invention includes an isolated peptide as disclosed herein for usein medicine; for use in therapy; and/or for use as a medicament. Forexample, the invention includes an isolated peptide as disclosed hereinfor use in regulating blood pressure-volume (for example in high bloodpressure or low blood pressure) and/or diuresis. The invention alsoincludes an isolated peptide as disclosed herein for use in treating aheart condition (for example heart failure).

According to a second aspect, the present invention provides an isolatedpeptide according to any aspect of the present invention for use inmedicine; for use in therapy; and/or for use as a medicament.

The invention includes the use of at least one isolated peptide asdisclosed herein in the preparation of a medicament for regulating bloodpressure-volume (for example in high blood pressure or low bloodpressure) and/or diuresis. The invention also includes the use of atleast one isolated peptide as disclosed herein in the preparation of amedicament for treating a heart condition (for example heart failure).

According to a third aspect, the present invention provides the use ofat least one isolated peptide according to any aspect of the presentinvention in the preparation of a medicament for regulating bloodpressure-volume and/or for treating a heart condition.

The invention includes a method of regulating blood pressure-volume (forexample in high blood pressure or low blood pressure) in a subjectcomprising administering an effective amount of an isolated peptide asdisclosed herein to the subject. The invention includes a method oftreating a heart condition (for example heart failure) in a subjectcomprising administering an effective amount of an isolated peptide asdisclosed herein to the subject.

According to a fourth aspect, the present invention provides a method ofregulating blood pressure-volume and/or treating a heart condition in asubject comprising administering an effective amount of an isolatedpeptide according to any according to any aspect of the presentinvention to the subject.

The pure vasodilator agents of the invention (such as DRG-K-Ring,GRD-K-Ring, DGD-K-Ring; SEQ ID NOs: 33-35) and DRD-ANP (SEQ ID NO: 51)may be used to help in “warm” and minimally “wet” patients in whom theneed is to redistribute blood volume from the heart and thorax to theperiphery and to avoid major volume depletion. These patients have wellpreserved BP and do not have extreme volume expansion and can toleratereductions in BP.

The diuretic only agents of the invention (such as DGD-ANP; SEQ ID NO:49) would be applied in volume expanded (edematous) patients with excessfluid in both lungs and periphery but low blood pressure. This “wet andcold” subgroup is very difficult to treat as there is a need to excreteexcess volume (sodium and water) whilst avoiding further reductions inBP which may precipitate worsening shock complicated by renal failure.

Compounds of the present invention will generally be administered as apharmaceutical formulation in admixture with a pharmaceuticallyacceptable adjuvant, diluent or carrier, which may be selected with dueregard to the intended route of administration and standardpharmaceutical practice. Such pharmaceutically acceptable carriers maybe chemically inert to the active compounds and may have no detrimentalside effects or toxicity under the conditions of use. Suitablepharmaceutical formulations may be found in, for example, Remington TheScience and Practice of Pharmacy, 19th ed., Mack Printing Company,Easton, Pa. (1995). For parenteral administration, a parenterallyacceptable aqueous solution may be employed, which is pyrogen free andhas requisite pH, isotonicity, and stability. Suitable solutions will bewell known to the skilled person, with numerous methods being describedin the literature. A brief review of methods of drug delivery may alsobe found in e.g. Langer, Science (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achievedroutinely by the skilled person using routine techniques and/or inaccordance with standard and/or accepted pharmaceutical practice.

The amount of a compound in any pharmaceutical formulation used inaccordance with the present invention will depend on various factors,such as the severity of the condition to be treated, the particularpatient to be treated, as well as the compound(s) which is/are employed.In any event, the amount of a compound in the formulation may bedetermined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule maycontain from 1 to 99% (w/w) active ingredient; from 0 to 99% (w/w)diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5%(w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50%(w/w) of a granulating agent or binder; from 0 to 5% (w/w) of anantioxidant; and from 0 to 5% (w/w) of a pigment. A controlled releasetablet may in addition contain from 0 to 90% (w/w) of arelease-controlling polymer.

A parenteral formulation (such as a solution or suspension for injectionor a solution for infusion) may contain from 1 to 50% (w/w) activeingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolidcarrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) ofone or more other excipients such as buffering agents, antioxidants,suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well asthe route of administration, compounds may be administered at varyingtherapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in thecontext of the present invention should be sufficient to effect atherapeutic response in the mammal over a reasonable timeframe. Oneskilled in the art will recognize that the selection of the exact doseand composition and the most appropriate delivery regimen will also beinfluenced by inter alia the pharmacological properties of theformulation, the nature and severity of the condition being treated, andthe physical condition and mental acuity of the recipient, as well asthe potency of the specific compound, the age, condition, body weight,sex and response of the patient to be treated, and the stage/severity ofthe disease.

Accordingly, in a preferred embodiment the invention includes anycomposition containing the given polynucleotide or amino acid sequenceof the invention.

The invention also includes an isolated nucleic acid molecule encodingan isolated peptide as disclosed herein.

According to a fifth aspect, the present invention provides an isolatednucleic acid molecule or polynucleotide encoding an isolated peptideaccording to any aspect of the present invention. Preferably the nucleicacid molecule or polynucleotide is comprised within an expressionconstruct to produce peptides of the invention.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

EXAMPLES

Standard molecular biology techniques known in the art and notspecifically described were generally followed as described in Green andSambrook, Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (2012).

Example 1 Materials and Methods Synthesis and Purification of ANP,K-Ring and Variants

All peptides were synthesized using manual Fmoc-based peptide synthesis.ANP and variants were synthesized using Tyr-preloaded Wang resin whileK-Ring and variants were synthesized using Novasyn TGR resin. Themethodology adopted for synthesis, purification and folding are asdescribed in Sridharan et al., [Biochem J 469 (2): 255-66 (2015)].

Animals

Male Sprague-Dawley (SD) rats (220-280 g) were obtained from Invivos,Singapore. Animals were acclimatized in the Animal Holding unit, NUS for3 days before the experiment. The experiments were conducted under theguidance and approval of Institutional Animal Care and Use Committee,NUS (041/12).

In-Vivo Activity Assay

The experiments were performed as described in Sridharan et al.,[Biochem J 469 (2): 255-66 (2015)]. In brief, animals were anesthetizedwith sodium pentobarbital (60-70 mg/kg) and catheters were inserted infemoral artery, vein and urinary bladder. The body temperature of theanimals was maintained using a thermostatic heat pad at 37° C.Continuous infusions (2 ml/h) of 0.2% BSA saline (with or withoutpeptide) was administered through the femoral vein while a pressuretransducer attached to the femoral artery recorded on-line changes inmean arterial pressure (MAP), heart rate (HR) and pulse pressure (PP).Urine was collected and the volume was measured after every 10 min.Experiments encompassed control (2×10 min), experimental (1×10 min) andrecovery (4×10 min) periods. All animals were euthanized at the end ofthe experiment in carbon-di-oxide chambers.

Cell Culture and Transfection

The experiments were performed as described in Sridharan et al.,[Biochem J 469 (2): 255-66 (2015)]. Dulbeco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100μg/ml streptomycin and 2 mM glutamine was used to grow CHO-K1 cells in ahumidified incubator at 37° C. with 5% CO₂. The cells were maintained bysub-culturing using 0.5% trypsin every three days. Cells (1×10⁵) wereseeded per well in 24-well plate and grown for 16 h. Cells in each wellwere transfected with 0.8 μg of plasmid encoding rat NPR-A using 2 μl ofLipofectamine™ 2000 transfection reagent. Cells were treated with thepeptides 24 h after transfection.

Whole Cell cGMP Assay

NPR-A transfected CHO-K1 cells were treated with varying concentrationsof peptides (10 nM to 10 μM dissolved in basal media containing 0.5 mMIBMX) for 30 min. The end-point accumulation of cGMP was measured aftercell-lysis with 200 μl of 0.1 N HCl using Enzo life sciences cGMP ELISAkit (manufacturer's protocol).

Time-chase study was performed by treating the cells with EC₅₀concentration of the peptides. The reactions were terminated at theindicated time-points (0, 2, 5, 10, 20 and 30 min) by treating the cellswith 0.1 N HCl. The cGMP concentration in the cell lysate was measuredusing cGMP ELISA kit (Enzo life sciences).

Statistical Analysis

The data are represented as mean±SEM. Statistical analysis was performedusing one way-ANOVA using two-tailed t-test. A p-value <0.5 wasconsidered significant.

Example 2 Functional Switches in K-Ring Responsible for HemodynamicEffects

To understand the role of unusual substitutions within K-Ring and theC-terminal tail, we evaluated pharmacological activities of severalmutants. The first generation of single mutants included substitution ofD3, R4 and D14 with G (FIG. 2A) within K-Ring. These single pointmutants, ^(GRD)K-Ring, ^(DGD)K-Ring and ^(DRG)K-Ring (the ‘mutated’residues are underlined), were synthesized (FIG. 5, Table 3) and theirin-vivo activities were evaluated in anesthetized rats (FIGS. 2B, 2C).Infusion of 2 nmol/kg/min of all the three single mutants inducedsimilar drop in MAP (≈12 mmHg) without any significant change in urinevolume (FIGS. 2B, 2C, Table 1) as K-Ring. ^(GRD)K-Ring evoked a greaterdrop on heart rate (HR) (−44.8±20.2 BPM) compared to K-Ring (−22.2±12.1BPM), which was similar to that of 0.2 nmol/kg/min infusion of ANP(−47.0±13.5 BPM) (FIGS. 6A, 6B, Table 1). Further this peptide alsoelicited greater reduction in pulse pressure (PP) (−11.5±2.3 mmHg)compared to both K-Ring (−4.5±1.7 mmHg) and ANP (−7.7±3.9 mmHg) (FIGS.5A, 5B, 6A, 6B, Table 1). ^(DGD)K-Ring infusion resulted in similar HRand PP changes (−21.8±9.5 BPM, −5.8±0.97 mmHg) as K-Ring (Table 1). Thismutant showed a slower initial decrease in BP and HR as the peptide wasinfused (FIGS. 6A, 6B). ^(DRG)K-Ring infusion, although showed similarvascular response (HR: −18.1±3.1 BPM and PP: −3.2±3.0 mmHg) like K-Ring(Table 1), but the changes in MAP and HR did not recover back to thebaseline within the experimental period (FIGS. 2B, 6A, 6B).Subsequently, we synthesized a double mutant, ^(GRG)K-Ring, in whichboth D residues were substituted by G. This peptide elicited similarchanges in MAP, HR and urine flow rate (MAP: −11.8±1.5 mmHg, HR:−39.1±11.5 BPM 11.4±3.8 μl/min). Despite the similar responses to ANP,^(GRG)K-Ring showed greater influence on PP (−12.9±0.7 mmHg) like^(GRD)K-Ring (FIGS. 2, 6A, 6B). Thus, the results indicated that the D3Gsubstitution led to a higher drop in HR and PP, for similar changes inMAP while D14G substitution delayed the recovery of MAP and HR.Interestingly, substitution of both D3 and D14 by G introduces renalactivity in K-Ring peptides. Thus, both D3 and D14 residues act as thecontrol switches that toggle the functions from a classical NP (bothrenal and vaso-active) to an only vasodilatory peptide.

To understand the role of a C-terminal tail in the in-vivo functions ofNPs, the C-terminal tail of K-Ring (RH) was replaced by that of ANP(NSFRY). This variant, K-Ring^(NSFRY), exhibited a≈15 mmHg drop in MAPfor a 2 nmol/kg/min infusion. It showed similar changes in HR and PP(−37.5±15.1 BPM and −7.3±0.7 mmHg; FIGS. 2B, 6A, 6B) as ANP along withdiuresis (12.3±3.8 μl/min). The peak of diuresis was delayed by 10 min(FIG. 2C). Thus, chimera K-Ring^(NSFRY) showed predominantcharacteristics of ANP with delayed renal response. Hence, the presenceof NSFRY tail appears to overcome the presence of D3, R4 and D14residues within the ring. Thus, the C-terminal tail seems to functionsas an alternate, but ‘forceful’ molecular switch which controls in-vivopharmacological effects. Thus, through systematic mutation studies, weidentified two functional switches for diuresis: two Gly residues withinthe ring and NSFRY tail. Using these switches we could manipulate thepharmacological effects of K-ring.

Example 3 Functional Switches are Transferable

To further understand the significance of these switches, wesystematically introduced Asp residues (replacing G3 and G14) and Argresidue (replacing G4) in ANP. A 2 nmol/kg/min infusion of ^(DGD)-ANPinduced marked increase in urine flow rate (7.9±2.1 μl/min) similar toANP (8.8±1.3 μl/min), but with much milder changes in MAP, HR and PP(−4.8±2.3 mmHg, −7.8±9 BPM and −4.3±1.4 mmHg, respectively) compared toanimals which received 0.2 nmol/kg/min of ANP (−10.1±0.8 mmHg,−47.1±13.5 BPM and −7.7±1.8 mmHg, respectively; FIGS. 3B, 3C, FIGS. 7A,7B, Table 1). The peak of diuresis with ^(DGD)ANP was delayed by 10 min,similar to K-Ring^(NSFRY). Thus, this variant has similar activityprofile as ANP; diuresis activity precedes hypotension, but theintroduction of D3 and D14 has widened the gap between the effectiveconcentrations of the peptide required to induce diuresis andvasodilation. In contrast, infusion of 2 nmol/kg/min of ^(DRD)ANPdisplayed potent vascular effects (−18.3±2.3 mmHg in MAP, −66.6±14.6 BPMin HR, −7.9±1.8 mmHg in PP) along with profound renal function (18.7±0.7μl/min urine flow rate) compared to ANP (FIGS. 3B, 3C, 8A, 8B, Table 1).At a lower dose (0.2 nmol/kg/min), ^(DRD)ANP manifested similarreduction in BP, HR and PP (−12.9±1.5 mmHg, −37.5±9.5 BPM, −5.8±2.8 mmHgrespectively) at an equimolar infusion of ANP, but failed to inducediuresis (Table 1). Thus, ^(DRD)ANP infusion manifested vasoactivityahead of renal function (FIGS. 3D, 3E, 8A, 8B). These observationssupport the role of D3 and D14 residues as functional switches. ResidueR4 along with D3 and D14 seems to reverse the in-vivo activities in adose-dependent manner; hypotension at low doses followed by bothvascular and renal activities with subsequent increment in concentrationof the peptide (FIGS. 3B, 3C). These results indicate that thefunctional switches identified in K-Ring are transferable to ANP.

Example 4 Stimulation of Guanylyl Cyclase Activity of NPR-A

Physiologically, ANP interacts with NPR-A and stimulates its guanylylcyclase activity to elevate the intracellular cGMP levels [Oliver, P.M.; et al., Proc Natl Acad Sci USA 94 (26): 14730-5 (1997)]. Previously,we showed that K-Ring is an NPR-A agonist with a 10-fold lower potencycompared to ANP [Sridharan, S.; Kini, R. M., Biochem J 469 (2): 255-66(2015)]. To understand the role of various residues in K-Ring and ANPmutants, we evaluated their ability to evoke cGMP response in CHO-K1cells transiently expressing rat NPR-A receptors (FIGS. 4A, 4B, Table2). Among the single point mutants of K-Ring, ^(GRD)K-Ring exhibited5-fold higher potency, while ^(DRG)K-Ring and ^(DGD)K-Ring exhibited a3- to 4-fold drop in potency compared to K-Ring. The double mutant^(GRG)K-Ring also exhibited 4-fold higher potency compared to K-Ring(FIG. 4A, Table 2). These results indicate that D3G substitution led toan increased potency, while the substitution at other positions led todecreased potency. In the double mutant, substitution of D3 led toincrease while the second substitution at D14 led to 20% loss inpotency. The introduction of ANP's C-terminal tail in K-Ring^(NSFRY)resulted in 5-fold improved activity. Thus, it is evident that thepresence of the first Gly residue at position 3 or the C-terminal tailof ANP improves the efficacy of receptor activation.

ANP mutants ^(DRD)ANP and ^(DGD)ANP exhibited 3-fold and 8-fold drop inactivity compared to ANP (FIG. 4B, Table 2). Both K-Ring^(NSFRY) and^(DRD)ANP elicited equipotent response. The difference in potenciesbetween ^(DDG)ANP and ^(DRD)ANP is similar to K-Ring and ^(DGD)K-Ring,implying that R↔G changes contribute equivalently in both NPs.

We also evaluated the activation kinetics of NPR-A in the presence ofthe mutant ANP and K-Ring to understand the dynamic changes in cGMPgeneration which would influence the spatio-temporal distribution of thesecondary messenger. A time-chase study of cGMP accumulation at EC₅₀concentrations of the peptides was assessed. ANP evoked an instantaneousincrease in cGMP level whereas K-Ring exhibited slower activationkinetics (2 min versus 30 min to produce similar levels of cGMP (FIGS.4C-4F). The rates of generation of cGMP by single point mutants^(DRG)K-Ring, ^(DGD)K-Ring were slower than K-Ring, whereas ^(GRD)K-Ringinduced a slightly increased rate of cGMP production. The double mutant^(GRG)K-Ring exhibited improved activation kinetics (3-fold) compared toK-Ring with the maximum accumulation of cGMP within 10 min. Although^(GRD)K-Ring and ^(GRG)K-Ring had comparable EC₅₀ concentrations theactivation kinetics was distinctly different (the peak of cGMPproduction was 10 min and 20 min respectively). K-Ring^(NSFRY) showedsimilar improvement in activation kinetics as ^(GRG)K-Ring; with a peakresponse at 10 min followed by a slow decay of cGMP to reach thesaturation value. The variants of ANP showed slower activation kineticscompared to the wild type; both ^(DRD)ANP and ^(DGD)ANP showed 2.6- and7.8-times slower kinetics (maximum cGMP elevation at 10 min). ^(DGD)ANPexhibits faster decay of intracellular cGMP compared to ^(DRD)ANP. Theseobservations suggest that D residues indeed influence receptor bindingand secondary messenger generation. The variants which exhibited fasteractivation kinetics seemed to show a decay of cGMP after the peakresponse. Since the cells were treated with phosphodiesterase inhibitor(0.5 mM IBMX), most likely the rate of degradation of cGMP wasnegligible. The treatment of the cells with EC₅₀ concentration of thepeptide ensured the cellular GTP was not limiting. Hence, the observeddecay in cGMP production in certain variants suggests that thesepeptides most likely caused desensitization of the receptor.

Overall, the activation kinetics of NPR-A clearly distinguishes variousNP mutants; vasoactive NPs exhibit slower kinetics compared vaso- andrenal active NPs exhibit relatively faster kinetics.

TABLE 1 Summary of in-vivo responses of ANP, K-Ring and their variantsMean arterial pressure Heart rate Pulse pressure Urine flow rate PeakPeak Peak Peak Peak Peak Peak Peak Peptide response time response timeresponse time response Time (nmol/kg/min) (mmHg) (min) Recovery (BPM)(min) Recovery (mmHg) (min) Recovery (μl/min) (min) Recovery ANP (0.08) −1.6 ± 1.5 — — −7.8 ± 2.4 — — −2.1 ± 1.5 — — 5.4 ± 1.4 20 Y ANP (0.2)−10.1 ± 1.9 30 Y −47.1 ± 13.5 30 Y −7.7 ± 3.9 40 Y 8.8 ± 1.3 20 Y K-Ring(2.0) −11.8 ± 2.4 30 Y −22.2 ± 12.1 20 Y −4.5 ± 1.7 20 — <2 — — K-Ring(10.0) −17.9 ± 2.2 30 N −24.9 ± 4.3  30 Y −5.6 ± 2.6 30 Y <2 — —^(GRD)K-Ring −11.3 ± 4.3 30 Y −44.8 ± 20.1 30 Y −11.4 ± 2.4  20 Y <2 — —(2.0) ^(DRG) K-Ring −10.5 ± 1.2 30 N −18.8 ± 3.2  40 N −3.2 ± 3.0 — — <2— — (2.0) ^(DGD)K-Ring −12.5 ± 2   30 Y −21.9 ± 9.8  30 Y −5.8 ± 0.9 30Y <2 — — (2.0) ^(GRG) K-Ring −11.6 ± 1.5 30 Y −39.0 ± 2.8  20 Y −12.9 ±0.8  30 Y 11.4 ± 3.8  20 Y (2.0) K-Ring ^(NSFRY) −15.1 ± 1.4 30 Y −37.4± 15.4 30 Y −7.3 ± 0.7 30 Y 10.8 ± 2.9  30 Y (2.0) ^(DGD) ANP  −4.8 ±2.9 — — −7.5 ± 8.9 — — −4.5 ± 1.4 — — 7.9 ± 2.0 30 Y (2.0) ^(DRD) AND−18.4 ± 2.4 30 Y −66.6 ± 14.5 30 N −8.0 ± 1.8 20 Y 18.8 ± 0.7  20 Y(2.0) ^(DRD) ANP −12.9 ± 1.5 30 Y −37.5 ± 9.5  30 Y −5.9 ± 2.0 20 Y <2 —— (0.2) Experiments consisted of six 10 min points. One control period(0-10 min), one infusion period (10-20 min), four recovery periods(10-30 min, 30-40 min, 40-50 min and 50-60 min). The end represented isthe average responses over the 10 min period from five independenttrials.

TABLE 2 Summary of receptor activation potency and kinetics of ANP,K-Ring and their variants Rate of NPR-A generation activation Ratio ofcGMP Ratio potency Ratio compared (pmol Ratio compared IC₅₀ comparedwild type cGMP/pmol compared wild type Peptide (nM) ANP NPs peptide) ANPNPs ANP 17.3 1 1 7.97 1 1 K-Ring 243.7 14.1 1 0.0228 349.6 1^(GRD)K-Ring 50.7 2.9 −4.8 0.113 70.5 5.0 ^(DRG) K-Ring 693.8 40.1 2.80.007 1138.6 3.2 ^(DGD)K-Ring 866.2 50.1 3.6 0.005 1594.0 4.6 ^(GRG)K-Ring 87.7 5.1 −2.8 0.19 41.9 8.3 K- 43.8 2.5 −5.6 0.31 25.7 13.6 Ring^(NSFRY) ^(DGD) ANP 134.9 7.8 7.8 0.14 56.9 56.9 ^(DRD) ANP 45.2 2.6 2.60.38 21.0 21.0

TABLE 3 Masses of ANP, K-Ring and their variants Calculated oxidizedmass Observed mass Peptide (Da) (Da) K-Ring 2769.1 2768.9 ± 0.4 ^(DRG)K-Ring 2711.1 2711.6 ± 0.3 ^(G) ^(RD)K-Ring 2711.1 2711.1 ± 0.4^(DGD)K-Ring 2670.2 2670.2 ± 0.3 ^(G) ^(RG) K-Ring 2653.0 2653.3 ± 0.2K-Ring ^(NSFRY) 3143.1 3143.2 ± 0.2 ANP 3080.4 3080.5 ± 0.5 ^(D) ^(GD)ANP 3196.2 3196.6 ± 0.3 ^(DRD) ANP 3277.6 3277.8 ± 0.2

TABLE 4 Rate of change of HR and PP per unit change in MAP of ANP,K-Ring and variants HR PP Rate of Correlation Rate of CorrelationPeptide change efficient change efficient K-Ring 1.7 0.66 0.5 0.67^(DRG) K-Ring 1.6 0.89 0.25 0.44 ^(G) ^(RD)K-Ring 2.82 0.93 0.7 0.8^(DGD)K-Ring 1.5 0.78 0.4 0.75 ^(G) ^(RG) K-Ring 3.1 0.80 1.2 0.9 K-Ring^(NSFRY) 2.4 0.92 0.42 0.75 ANP* 5.6 0.95 0.5 0.65 ^(D) ^(GD) ANP 4.40.44 0.5 0.4 ^(DRD) ANP 4.5 0.95 0.5 0.6 The data represented in thecorrelation between MAP and other vascular parameter for an infusion of2 nmol/kg/min of peptide except ANP (0.2 nmol/kg/min)

DISCUSSION

The antihypertensive and anti-hypervolemic properties of ANP areconcentration-dependent; exclusive renal functions manifest at a lowerdose (0.02-0.1 nmol/kg/min) and both the activities are exhibited atslightly higher doses (>0.1 nmol/kg/min) [Morice, A.; et al., Clin Sci(Lond) 74 (4): 359-63 (1988)]. Thus, the differences in the thresholdconcentrations that segregate the activity on kidney and circulation areless than 5-fold apart and are variable among different studies[Soejima, H.; et al., Am J Physiol 255 (3 Pt 2): R449-55 (1988); Morice,A.; et al., Clin Sci (Lond) 74 (4): 359-63 (1988)]. Although this dosedependency may be attributed to the NPR-A expression levels in thetarget tissues (kidney>blood vessels) [Uhlen, M.; et al., Science 347(6220): 1260419 (2015)], the exact molecular mechanisms are unclear.Structure-activity studies of ANP have identified vital residues (F8,M13, D14, and R15 within the ring and the C-terminal NSFRY) which arenecessary for NPR-A binding [Olins, G. M.; et al., J Biol Chem 263 (22):10989-93 (1988)] and conferring the physiological activity. Severalstudies have utilized phage display to increase the specificity of ANPvariants to NPR-A and improve the natriuretic/diuretic response [Jin,H.; Li, B.; et al., J Clin Invest 98 (4): 969-76 (1996)]. However, themolecular determinants of vascular and renal function in NPs remainunidentified.

Recently we identified and characterized an exogenous NP from kraitvenom (KNP) [Sridharan, S.; Kini, R. M., Biochem J 469 (2): 255-66(2015)]. This peptide has the conserved NP ring with a 38-residue longC-terminal tail (FIG. 1A) which had propensity to form α-helix. Ourstructure-function studies showed that KNP has two pharmacophores:K-Ring and Helix. These functional segments induced vasodilation throughorthogonal pathways. K-Ring, like a classical NP, elevates intracellularcGMP levels through activation of NPR-A with a 10-fold lower potencycompared to ANP, while Helix uses NO-dependent mechanisms [Sridharan,S.; Kini, R. M., Biochem J 469 (2): 255-66 (2015)].

Further, we observed that the infusion of 2 nmol/kg/min of K-Ring inanesthetized rats showed a 12 mmHg drop in mean arterial pressure (MAP)(recovered to baseline within experimental period) without significantchanges in urine volume [Sridharan, S.; Kini, R. M., Biochem J 469 (2):255-66 (2015)]. A 5-times higher dose of K-Ring showed further drop inMAP (−19.1±2.5 mmHg with no recovery) without affecting urine volume(FIGS. 1B, 1C). Thus, K-ring induced vasodilation with minimal or nodiuretic effects. This is in contrast to ANP's ability to inducediuretic and vasodilatory activities; ANP at a low dose (0.08nmol/kg/min) showed increased urine volume without any alteration in MAPwhile a slightly higher dose (0.2 nmol/kg/min) showed both reduction inMAP along with diuresis (FIGS. 1B, 1C), as was observed previously[Soejima, H.; et al., Am J Physiol 255 (3 Pt 2): R449-55 (1988)]. Thusthe observed difference in the in-vivo activity of these peptidessuggested that, K-Ring has distinct hemodynamic and diuretic effectscompared to ANP and it could serve as a molecular roadmap to delineatethe determinants of hypotensive and diuretic functions in NPs.

Structure-activity studies on ANP have shown that certain conservedresidues (F2, M6, D7, R8, 19 and L15) within the 17-membered ring andits C-terminal tail (NSFRY) are crucial for NPR-A binding and in-vivoactivity [Li, B.; et al., Science 270 (5242): 1657-60 (1995); Olins, G.M.; et al., J Biol Chem 263 (22): 10989-93 (1988); Ogawa, H.; et al., JBiol Chem 279 (27): 28625-31 (2004)]. All critical residues necessaryfor receptor binding within the ring are conserved in K-Ring, but itsC-terminal tail has only two residues (RH). In comparison to ANP, K-Ringhas several substitutions within the ring (G3D, G4R, G9S, A10T, Q11H andG14D). Among these, residues at positions 9, 10 and 11 are variableamong several NPs (FIG. 1A). Thus, we hypothesized that the distinctstructural differences, G3D, G4R and G14D substitutions along withshorter C-terminal tail, may be responsible for the observed differencesin the hemodynamic and diuretic effects of ANP and K-Ring. In thepresent study, we address their role in imparting the in-vivo functionsof NPs using systematic substitution. Further, using decipheredstructural details we have engineered ANP analogues with either vascularor renal functions. Such variants with exclusive functions have immensevalue as therapeutic agents in treating heart failure patients. Thesestructure-function relationship studies also identify the key residuesthat impact on NP-induced vasodilation, heart rate and diuresis.

K-Ring Helps in Delineation of Vasodilatory and Diuretic Effects

Our previous study showed that K-Ring (70% identical to ANP) exclusivelyinduced hypotensive effects without altering renal output, despite beingan NPR-A agonist [Sridharan, S.; Kini, R. M., Biochem J 469 (2): 255-66(2015)]. This peptide failed to evoke renal response even at 100-foldhigher concentrations than ANP that induced exclusive diuresis (FIGS.1B, 1C). Hence, K-Ring structure seemed to encode the molecularinformation that governs tissue-specific responses of NPs and thus,provided impetus to identify the residues, which play critical role indetermining selectivity towards vascular and/or renal functions and actas functional switches.

Role of G3:

G3 is conserved in almost all NPs. But this residue is replaced by Aspin K-ring (FIG. 1A). Our results suggest that G3 is a crucial residuewithin the ring of NP which imparts potent vascular functions. Thesubstitution of this residue with negatively charged Asp decreased thepotency to activate NPR-A by ˜3- to 10-fold (FIG. 4A, B, Table 2) andlowered the activation kinetics (5-fold) (FIG. 4C, D, Table 2). G3Dsubstitution also seemed to lower the influence on HR and PP, forsimilar BP changes (FIG. 2A, 6A, B); D3 variants showed smaller changein HR and PP per mm Hg drop in BP (1.5 times and 0.3 times,respectively) compared to G3 variants (3.0 times and 0.8 times,respectively) (Table 4). Thus, G3↔D3 changes acted as the molecularswitches which controlled the HR and PP changes of a NP.

Role of G14:

Within K-Ring scaffold, the substitution of D14 to G appeared to lowerthe potency of NPR-A by 10-fold (Table 2) and activation kinetics by3-fold (FIG. 4C). This variation at positon 14 seemed to sustain thechange in BP and HR all throughout the experimental period with minimalrecovery. Hence, it may be suggested that G14 is key residue necessaryto elicit sustained vascular activity. In comparison between^(GRG)K-Ring, ^(DRG)K-Ring and ^(GRD)K-Ring, it is evident that G14controlled the renal function of a NP. The presence of both G3 and G14are necessary to warrant renal functions to NPs.

The residues D3 and D14 seem to lower ability of a NP to evoke cGMPresponse. From the crystal structure of ANP-NPR-A complex, G3 and G14seem to be in close proximity with a negatively charged pocket (E169^(A)and E169^(B) of receptor subunits) [Ogawa, H.; et al., J Biol Chem 279(27): 28625-31 (2004)]. The presence of D in the place G could introduceconformational constraint as well as electrostatic repulsion between thenegatively charged pocket of NPR-A, which might disengage key molecularinteractions and mediate activation through a varied set of structuralframework compared to high affinity native ligands. The differences inthe conformational selectivity imposed on the extracellular domain bydifferent ligands may alter the relay of allosteric activation ofintracellular guanylcyclase domain thereby leading to distinctiveactivation kinetics. Previous studies have shown that the mutations ofG3A and G14A did not alter the binding efficiency significantly,suggesting that the loss of conformational flexibility in the absence ofGly residues had no influence [Li, B.; et al., Science 270 (5242):1657-60 (1995); Watanabe, T. X.; et al., Eur J Pharmacol 147 (1): 49-57(1988)]. Thus, our results indicate that the incorporation of Aspresidues at 3 and 14 and introduction of bulkier and charged side chainmight lower the ligand's ability to activate NPR-A and influence itsin-vivo activity.

Role of G4:

Substitution of G4 to Arg seemed to improve the NPR-A activation potencyby 3-fold. The difference in activity was observed between ^(DGD)K-Ringand K-Ring as well as ^(DGD)-ANP and ^(DRD)ANP suggesting that R4 may beimportant to compensate the disengagement of interaction with NPR-A dueto the introduction of negatively charged residues in both thescaffolds. This residue seems to be important for reversal of functionselectivity (vasoactivity followed by renal) in ANP frame work.^(DGD)K-Ring induces a slower rate of BP decrease compared to^(DRD)K-Ring, suggesting the lower influence on the system (FIGS. 2B,2C). The NP variants with G3 and G14 exhibited 10-fold improved NPR-Aactivation potency and 3-fold faster kinetics compared to the variantswith Asp in these positions. Both G3 and G14 seemed to be the necessaryto display renal activity in K-Ring scaffold and hence identified as‘diuretic switch residues’.

Role of the C-Terminal NSFRY:

The C-terminal NSFRY acts as the alternate, but ‘forceful’ diureticfunctional switch which introduces renal function in peptides which areexclusively hypotensive. Addition of C-terminal tail to K-Ring improvesNPR-A activation potency (5-fold) and kinetics (10-fold). The crystalstructure of ANP-NPR-A shows residues N, S, F, R of the C-terminal tailform hydrogen bonding and pi-pi interaction with Q186^(B), E187^(B) andF188^(B) of the receptor. Earlier mutagenesis studies also revealed thatthe absence of the C-terminal tail decreases the ability to elicitvasorelaxation (30-fold) and natriuresis (3-fold) of ANP [Watanabe, T.X.; et al., Eur J Pharmacol 147 (1): 49-57 (1988)]. Thus C-terminal tailappears to be an important molecular switch. The presence of theC-terminal tail increases the influence on HR and PP (FIGS. 3A-3C,7A-7D). Thus, the addition of C-terminal tail improved the vascularactivity and imparted renal functions. Although both ^(DRD)ANP andK-Ring^(NSFRY) elicited equipotent activation potency and kinetics ofNPR-A, equimolar infusion of these peptides resulted in ^(DRD)ANPexhibiting influence on HR (2-fold) and diuresis (2-fold) compared toK-Ring^(NSFRY) (Table 1).

The inclusion of NSFRY as C-terminal tail in K-Ring scaffold showed adelayed diuresis peak compared to ^(DRD)ANP (FIG. 3C). These differencesin the in-vivo effects may be attributed to the structural differencesin positions 9, 10 and 11 (G, A, Q in ANP versus S, H, T in K-Ring)between the two NPs. A previous study using phage display library showedthat G9R, A10E and Q11A mutations seemed to improve natriuretic anddiuretic functions [Cunningham, B. C.; et al., EMBO J 13 (11): 2508-15(1994)]. Thus, further studies are needed to evaluate the roles of theseresidues in the renal functions of an NP.

NPR-A Receptor Activation Kinetics and Function

The differences in the pharmacological functions of NP variants may beexplained through: (a) expression level of NPR-A in different targettissues; (b) expression pattern of clearance receptor (NPR-C); (c)binding and activation kinetics of the ligand; and (d)compartmentalization of signals. Gene expression levels andhistochemical staining experiments reveal higher levels of NPR-Aexpression in kidney compared to vascular smooth muscle [Uhlen, M.; etal., Science 347 (6220): 1260419 (2015)]. Although NPR-C expressionfollows a similar trend (expression higher in kidney compared tovascular smooth muscle) the ratios of NPR-A and NPR-C in these tissues,which determines the activation profile, are different [Uhlen, M.; etal., Science 347 (6220): 1260419 (2015)]. The onset of renal functionsat low doses of ANP may be attributed to the differential in theexpression pattern of receptors in the target tissue. All NP analoguesactivated NPR-A but their activation kinetics varied significantly(Table 2). The data show that (i) exclusively hypotensive peptidesexhibited slow activation kinetics compared to both diuretic andvasodilatory peptides and (ii) peptides exhibiting faster activationshowed decay of cGMP signal after a peak response. This difference inthe rate of synthesis of cGMP would alter the diffusion rates, whichmight introduce differential spatio-temporal distribution andcompartmentalization of the signal to activate only certain downstreameffectors leading to selective downstream function [Piggott, L. A.; etal., J Gen Physiol 128 (1): 3-14 (2006)]. The key downstream effectorsin both cell types are protein kinase G (cytosolic), phosphodiesterase(both cytosolic and membrane associated) and cyclic nucleotide gated ionchannels (membrane bound) [Potter, L. R.; et al., Handb Exp Pharmacol(191): 341-66 (2009)]. The differential localization of these proteinsnecessitates the diffusion of the secondary messenger to initiate thesignaling cascade. There is substantial evidence enunciating the role ofphosphodiesterases in compartmentalizing cyclic nucleotide signals[Fischmeister, R.; et al., Circ Res 99 (8): 816-28 (2006)] and this 2006study suggests that diffusion enhanced spatial segregation of signalcontours may aid in attributing specific downstream functions. Thus,further characterization of the differences in intracellular signalsimposed by the native ligands (ANP and BNP) or NP analogues describedhere may help us understand how these peptides exhibit distinctphysiological or pharmacological profiles.

Delinking Vasodilatory and Diuretic Functions of NPs

Our study delineated the salient features of NPs and identified themolecular functional switches that determine vasodilatory and/ordiuretic functions of NPs. Asp residues at position 3 and 14 in theabsence of the C-terminal tail are the key salient feature to developonly vasoactive NPs. Residue G3 seems to cause greater drop in HR andPP, and its substitution with Asp lowers the reduction of theseparameters for a similar change in MAP. The presence of G3 and G14 orthe presence of the C-terminal tail leads to diuretic functions. Asmentioned above, HF scenarios need personalized medicine depending onpressure-volume overload. These functional switches help create NPswhich are adjustable to the personalized treatments of HF patients.Thus, this study presents the design of potential NP analogues whichcould help in the development key therapeutic molecules. The presentstudy investigates the pharmacological roles of these peptides inanesthetized rats.

In conclusion, this study has pinpointed the molecular switches whichcontrol the activity of NPs on smooth muscle and kidney. This studywidens the scope for design of ligands for specific needs and provides aplatform to explore the receptor-effector system of NPR-A. Theengineered NP analogues may serve as key therapeutic leads that couldhelp regulate either blood pressure or volume in distinct cohorts of HFpatients.

SUMMARY

Natriuretic peptides have crucial effects in restoring the equilibriumduring heart failure. ANP elicits a concentration-dependent diureticand/or hypotensive functions; at low doses it elicits only renalfunction and at slightly higher doses it elicits both vascular and renalfunctions. K-Ring, the conserved ring of krait venom NP, exhibitedexclusive vasodilatory effects without altering urine volume at100-times the concentration of ANP. Here, we have delineated themolecular switches that control its vasodilatory and diuretic functionsthrough systematic substitution of residues at positions 3, 4 and 14within the ring and C-terminal tail. Infusion of various NP analogues inanesthetized rats indicated that

(a) G3 analogues significantly reduced the heart rate and pulse pressurecompared to D3 analogues;(b) G4 analogues exhibited exclusive diuresis at low dose andhypotension along with diuresis at higher dose while R4 analogueselicited a reversed preference of pharmacological activity;(c) G14 analogues showed sustained vasodilatory effects compared to D14analogues;(d) analogues with both G3 and G14 substitutions elicited diuresis,while D3 and D14 analogues exhibited exclusive hypotension withoutsignificant diuresis, thereby acting as ‘vasodilatory-diuresis toggle’switch; and(e) the analogue with the C-terminal tail (NSFRY) overrides theinfluence of D3 and D14 residues to induce diuresis in otherwise onlyvasodilatory peptides, thereby acting as ‘forceful diuresis’ switch.NPR-A activation potencies and kinetics of these peptides distinguishthe observed pharmacological profile; partial agonists with slowactivation kinetics exhibited only vasodilatory properties, while thosewith faster kinetics elicited both vasodilatory and diuretic effects.

REFERENCES

Any listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat such document is part of the state of the art or is common generalknowledge.

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1. An isolated peptide having vasodilatory and/or diuretic activity inmammals, comprising the amino acid sequence CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC(SEQ ID NO: 1), wherein X₁, X₂, X₈ each independently comprises anyamino acid; X₃ comprises R or K; X₄ comprises M, I or L; X₅ comprises G,S or N; X₆ comprises A, H or S; X₇ comprises Q, T, S, M or V; and X₉comprises I or L, wherein the peptide does not consist of the amino acidsequence depicted by SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55 or SEQ ID NO:
 56. 2. The isolated peptide of claim 1, comprisingan amino acid sequence selected from the group consisting of: (i)CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC (SEQ ID NO: 2) wherein X₁ comprises D or G;(ii) CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC (SEQ ID NO: 3) wherein X₂ comprises R orG; (iii) CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC (SEQ ID NO: 4), wherein X₈ comprisesD or G; (iv) (SEQ ID NO: 5) CFDX₂X₃X₄DRIX₅X₆X₇SX₈X₉GC; (v) (SEQ ID NO:6) CFGX₂X₃X₄DRIX₅X₆X₇SX₈X₉GC; (vi) (SEQ ID NO: 7)CFX₁RX₃X₄DRIX₅X₆X₇SX₈X₉GC; (vii) (SEQ ID NO: 8)CFX₁GX₃X₄DRIX₅X₆X₇SX₈X₉GC; (viii) (SEQ ID NO: 9)CFX₁X₂X₃X₄DRIX₅X₆X₇SDX₉GC; (ix) (SEQ ID NO: 10)CFX₁X₂X₃X₄DRIX₅X₆X₇SGX₉GC; (x) (SEQ ID NO: 11) CFDRX₃X₄DRIX₅X₆X₇SX₈X₉GC;(xi) (SEQ ID NO: 12) CFGRX₃X₄DRIX₅X₆X₇SX₈X₉GC; (xii) (SEQ ID NO: 13)CFDGX₃X₄DRIX₅X₆X₇SX₈X₉GC; (xiii) (SEQ ID NO: 14)CFGGX₃X₄DRIX₅X₆X₇SX₈X₉GC; (xiv) (SEQ ID NO: 15)CFDX₂X₃X₄DRIX₅X₆X₇SDX₉GC; (xv) (SEQ ID NO: 16) CFGX₂X₃X₄DRIX₅X₆X₇SDX₉GC;(xvi) (SEQ ID NO: 17) CFDX₂X₃X₄DRIX₅X₆X₇SGX₉GC; (xvii) (SEQ ID NO: 18)CFGX₂X₃X₄DRIX₅X₆X₇SGX₉GC; (xviii) (SEQ ID NO: 19)CFX₁RX₃X₄DRIX₅X₆X₇SDX₉GC; (xix) (SEQ ID NO: 20) CFX₁RX₃X₄DRIX₅X₆X₇SGX₉GC; (xx) (SEQ ID NO: 21) CFX₁G X₃X₄DRIX₅X₆X₇SDX₉GC, and(xxi) (SEQ ID NO: 22) CFX₁G X₃X₄DRIX₅X₆X₇SGX₉GC.

3-22. (canceled)
 23. The isolated peptide of claim 1, comprising theamino acid sequence CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GC (SEQ ID NO: 1), whereinX₁ is G or D, X₂ is G or R and X₈ is G or D.
 24. The isolated peptide ofclaim 1 comprising CFX₁X₂X₃X₄DRIX₅X₆X₇SX₈X₉GCNSFRY (SEQ ID NO: 23). 25.The isolated peptide of claim 1, wherein the peptide comprises asequence selected from the group consisting of: (SEQ ID NO: 24)CFDRRIDRISHTSDIGC; (SEQ ID NO: 25) CFGRRIDRISHTSDIGC; (SEQ ID NO: 26)CFDGRIDRISHTSDIGC; (SEQ ID NO: 27) CFDRRIDRISHTSGIGC; (SEQ ID NO: 28)CFGGRIDRISHTSDIGC; (SEQ ID NO: 29) CFGRRIDRISHTSGIGC; (SEQ ID NO: 30)CFDGRIDRISHTSGIGC; and (SEQ ID NO: 31) CFGGRIDRISHTSGIGC.


26. The isolated peptide of claim 1, wherein the peptide is selectedfrom the group consisting of: (SEQ ID NO: 32) GLLISCFDRRIDRISHTSDIGCRH;(SEQ ID NO: 33) GLLISCFDRRIDRISHTSGIGCRH; (SEQ ID NO: 34)GLLISCFGRRIDRISHTSDIGCRH; (SEQ ID NO: 35) GLLISCFDGRIDRISHTSDIGCRH; (SEQID NO: 36) GLLISCFGRRIDRISHTSGIGCRH; (SEQ ID NO: 37)GLLISCFDRRIDRISHTSDIGCNSFRY; (SEQ ID NO: 38)GLLISCFGRRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDL RADSKQSRAA; (SEQ IDNO: 39) GLLISCFDGRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDL RADSKQSRAA;(SEQ ID NO: 40) GLLISCFDRRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA; (SEQ ID NO: 41)GLLISCFGGRIDRISHTSDIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDL RADSKQSRAA; (SEQ IDNO: 42) GLLISCFGRRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDL RADSKQSRAA;(SEQ ID NO: 43) GLLISCFDGRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDLRADSKQSRAA; and (SEQ ID NO: 44)GLLISCFGGRIDRISHTSGIGCRHRKDPPRAPPAAPSAAPLAVTWLIRDL RADSKQSRAA.


27. The isolated peptide of claim 1, wherein the peptide is selectedfrom the group consisting of: (SEQ ID NO: 45)SLRRSSCFDGRMERIGAQSGLGCNSFRY; (SEQ ID NO: 46)SLRRSSCFGRRMDRIGAQSGLGCNSFRY; (SEQ ID NO: 47)SLRRSSCFGGRMDRIGAQSDLGCNSFRY; (SEQ ID NO: 48)SLRRSSCFDRRMDRIGAQSGLGCNSFRY; (SEQ ID NO: 49)SLRRSSCFDGRMDRIGAQSDLGCNSFRY; (SEQ ID NO: 50)SLRRSSCFGRRMDRIGAQSDLGCNSFRY; and (SEQ ID NO: 51)SLRRSSCFDRRMDRIGAQSDLGCNSFRY.


28. An isolated peptide of claim 1, wherein when X₁ is G and X₈ is G, orwhen NSFRY is present at the C-terminal end of the peptide, diureticactivity of the peptide is elicited.
 29. An isolated peptide of claim 1,wherein: a) when X₁ is G the peptide elicits reduced heart rate andpulse pressure compared to when X₁ is D; b) when X₂ is G the peptideelicits exclusive diuresis at low dose and hypotension along withdiuresis at higher dose; c) when X₂ is R the peptide elicits a reversedpreference of pharmacological activity to that shown in b); d) when X₈is G the peptide elicits sustained vasodilatory effects compared to whenX₈ is D; and e) when X₁ is D and X₈ is D the peptide elicits exclusivehypotension without significant diuresis.
 30. An isolated peptide ofclaim 29, wherein a peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 51 hasvasodilatory activity; a peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 36 andSEQ ID NO: 37 has vasodilatory and diuretic activity; and a peptidecomprising the amino acid sequence set forth in SEQ ID NO: 49 has renalactivity.
 31. An isolated peptide of claim 1 for use in medicine; foruse in therapy; and/or for use as a medicament.
 32. (canceled)
 33. Amethod of regulating blood pressure-volume and/or treating a heartcondition in a subject comprising administering an effective amount ofthe isolated peptide of claim 1 to the subject.
 34. An isolatedpolynucleotide encoding the isolated peptide of claim
 1. 35. Acomposition comprising the isolated peptide of claim
 1. 36. Acomposition comprising the isolated peptide of claim 31.